Cryobiology is the study of the effects of low temperatures on biological systems. Although freezing is lethal to most living systems, cryobiologists have been able to preserve cells and tissues at a range of subzero temperatures, as low as liquid nitrogen temperatures (−196° C.). Currently, cryoprotection can be applied to most cells in suspension, such as stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova, and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, cells of opthalmolic origin, cells of orthopedic origin, cells from connective tissues, cells of reproductive origin, and cells of cardiac origin). Cryopreservation has also been used to effectively preserve tissue, such as heart valves, embryos, skin, articular cartilage, and islets of Langerhans and an increasing range of engineered tissues and tissue constructs. Although the current recovery of viable cells post-thaw may be sufficient for some clinical uses, recovery is generally considered less than optimal due to injury during the freezing process.
Cryopreservation has been applied to many cell and tissue types. Recent developments in the utilization of a variety of stem cells, including umbilical cord blood stem cells have revived interest in optimizing cryopreservation techniques for cells and tissues (D. Krause, 2002). In particular, for stem cells and other cell types which are obtained in low numbers from donors, high recovery of these cell types is crucial. High recovery is also important in cryopreservation of engineered cells due to the high cost and length of time for manufacturing such cells. Emerging higher standards for cell and tissue banking (Guide to safety and quality assurance for organs, tissues and cells, 2nd edition, 2004, Council of Europe Publishing, France), specifically stem cell banking, will be required to meet future needs of cell banking and therefore, optimal cryopreservation techniques are fundamental.
Currently, cryopreservation of cells has been most successful with the use of cryoprotectants and cryopreservation of stem cells has been most successful with the use of the permeating cryoprotectant, dimethyl sulfoxide (DMSO). There are, however, limitations to the use of DMSO. Adverse affects have been associated with infusion of stem cells preserved with DMSO (Davis et al., 1990; Egorin et al., 2001; Santos et al., 2003; Zambelli et al., 1998). Some researchers have attempted to reduce the amount of DMSO (Abrahamsen et al., 2002; Beaujean et al., 1998) or combine it with a non-permeating cryoprotectant, such as Hydroxyethyl starch (HES) (Donaldson, 1996; Halle et al., 2001; Katayama et al., 1997).
In non-clinical studies to examine the effects of low temperatures on cells, some cells have been cryopreserved without the use of a specific cryoprotectant such as DMSO (Farrant et al, 1974; Knight, Farrant et al. 1977). However, cooling profiles were not optimized and cell recoveries were not as high as with cryoprotectants. These studies were used in research to understand the mechanisms of cryoinjury and cryoprotection.
In current cryopreservation procedures, cells are generally cooled at a constant rate which is optimized for the cell type and cryoprotectant. This protocol has typically been approached empirically by varying cooling rates and the nature and concentration of cryoprotectants. In addition to cooling at a constant rate, other techniques have been described to examine the effects of low temperatures on cells, including a two-step freezing technique. The two-step freezing technique (J. Farrant et al., 1974) is a method to examine the effects of osmotic interactions on cell recovery over a broad range of subzero temperatures. In this procedure, lymphocytes were cooled rapidly to various subzero temperatures and held for various periods of time before being 1) thawed directly from that holding temperature or 2) rapidly cooled to −196° C. before thawing. McGann and Farrant later reported that the subzero temperature and the length of hold time at that temperature were factors to consider when attempting to maximize cell survival (McGann and Farrant, 1976). To date an easy method that can optimize cooling profiles for a cell type or for various cell types for cryopreservation of cells is not available and a reliable method that does not use cryoprotectants, especially permeating cryoprotectants, has not been recommended, especially for clinical use of the cells.
There is significant interest in designing an optimized cryopreservation protocol for all cell types and tissues, which maintains cell and tissue viability but does not require toxic cryoprotectants. Further there is a need for protocols to cyropreserve larger volumes of cells and tissues. Further there is a need to develop a model or optimization protocol to optimize cooling profiles to cryopreserve cells.